JP2759656B2 - Method for producing useful substances by plant tissue culture - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing useful substances from plants.

(Prior Art) Conventionally, human beings have used natural organic compounds produced by plants, such as alkaloids, terpenoids, and pigments, as medicines and foods. When obtaining useful substances produced by these plants,
Extraction methods from naturally grown or cultivated plants have been adopted. However, the quantity and quality of plants depend on natural conditions, and it takes a lot of time and effort to harvest, and it is not an effective method for obtaining a large amount of useful substances. In recent years, useful substances have been systematically and stably produced by culturing a large amount of plant culture cells using a plant tissue culture technique.

Generally, in plant tissue culture, a part of a tissue piece is planted in a medium containing components necessary for plant growth, and indefinite cells called callus are induced and cultured indefinitely. At this time, in addition to inorganic salts, vitamins, sugars, and the like, plant hormones (known as auxins, gibberellins, cytokinins, ethylene, and the like) are added to the medium. Actually, production conditions of a useful substance using a plant tissue culture technique have been carried out by examining culture conditions such as light and temperature, a basic medium, a plant hormone, and the like in detail.

(Problems to be Solved by the Invention) In plant tissue culture for the purpose of producing a useful substance, it is desirable to rapidly and cultivate a large amount and rapidly of cultured cells containing a large amount of a useful substance. ,
The production of useful substances may be stopped at all, or the production amount may be greatly reduced. For tissue culture of plants, the addition of plant hormones is indispensable, but depending on the amount added, they may not only inhibit the production of useful substances but also inhibit the growth of callus. In general, the relationship between growth and metabolism is inconsistent with the growth rate of metabolism of useful substances. There are many problems. As described above, there are various problems in industrially producing a target useful substance using a plant tissue culture technique.

(Means for Solving the Problems) The present invention has been made in view of the problems described above, and when producing a useful substance produced by a plant by using a plant tissue culture technique, a photosynthetic prokaryotic microorganism culture filtrate and / or A gist of the present invention is a method for producing a useful substance, comprising using a plant tissue culture medium containing a photosynthetic prokaryotic microorganism extract.

Examples of photosynthetic prokaryotic microorganisms usable in the present invention include cyanobacteria (R. Rippka and RYStanier: J. Gen. M.
icobiol., 111 , 1-61 (1979) ". ) And photosynthetic bacteria. Examples of cyanobacteria include the genus Chlorogloe (Chlorogloe)
opisis sp., Dermocarpa sp., Nostoc sp., Synechococcus (Synech)
ococcus sp.), Oscillatoria s
p.), and specific examples include Chlorogloeopisis sp. ATCC 27181, Dermocarpa sp. ATCC 2937, and Nostock sp.
sp.) ATCC 27895, Synechococcus sp.
p.) ATCC 27192, ATCC 29404, ATCC 29534, ATCC 2717
0, Oscillatoria sp. ATCC 27906
The photosynthetic bacteria include, for example, genus Halobacterium (Halobacterium sp.), Genus Rhodopseudomonas sp., Genus Rhodospirillum (Rhodospirillum sp.). Halobacterium
cutirubrum) ATCC 33170, Halobacterium mediterranei ATCC 33500, Halobacterium saccum
harovorum) ATCC 29252, Halobacterium salinarium ATCC 19700, Halobacterium sodomense A
TCC 33755, Rhodoseumonas acidophila (Rhodo
pseudomonas acidophila) ATCC 25092, Rhodopseudomonas rutila ATCC 3387
2. Rhodopseudomonas spheroides
monas spheroides) ATCC 17024, Rhodoseudomonas
Viridis (Rhodopseudomonas viridis) ATCC 19567,
Rhodopseudomonas
blastica) ATCC 33485, Rhodospirillum molischianum ATCC 14031, Rhodospirillum photometric (Rhodospirillum photome)
tricum) ATCC 27871, Rhodospirillum rubrum (Rhod
ospirillum rubrum) ATCC 277, ATCC 17031, Rhodospirillum tenue ATCC 25093 and the like. The photosynthetic prokaryotic microorganisms are not limited to the above-mentioned microorganisms or their variants or mutants, and include marine and freshwater photosynthetic prokaryotic microorganisms isolated from nature.

Cultivation of photosynthetic prokaryotic microorganisms can be performed by culturing the photosynthetic prokaryotic microorganisms, usually using a medium containing inorganic salts or the like, in a tank culture, or in a culture such as outdoor open culture using sunlight. If the photosynthetic prokaryotic microorganism is naturally present in abundance to some extent, seawater or freshwater in which the microorganism grows can be used as the culture solution.

The photosynthetic prokaryotic microorganism culture filtrate can be obtained by centrifuging the culture solution obtained by the above-described culture method, or by performing filtration, etc.If the biological activity of the target culture filtrate is weak, then the filtrate is concentrated under reduced pressure. It may be used after being concentrated. At this time, if the concentration ratio is increased and the salt concentration is increased, the plant tissue may be adversely affected. Therefore, it is desirable to desalinate and use the plant tissue by electrodialysis or the like until there is no adverse effect. The extract of the photosynthetic prokaryotic microorganism is obtained by contacting the cells of the photosynthetic prokaryotic microorganisms or the appropriately crushed cells with an appropriate solvent at room temperature or heated. as,
Various solvents may be used alone or in combination depending on the cells, but aqueous solvents are generally preferred. For example, the aqueous solvent includes water alone or a solution in which an acid, a base, a salt, or an organic solvent is dissolved. After extraction with an organic solvent such as methanol, ethanol, ethyl acetate or ether, the organic solvent may be removed and then dissolved in water. Extracts that can be used in the present invention include extracts obtained by the above-described methods and fractions obtained from these extracts, and these can be appropriately concentrated or diluted before use. Furthermore, these extracts and fractions can be dried and used as a powder by drying under reduced pressure, freeze drying, spray drying and the like.

The amount of the photosynthetic prokaryotic microorganism culture filtrate and / or the photosynthetic prokaryotic microorganism extract added to the basic medium is 0.0001 to 50%, which can be appropriately selected depending on the purpose of use and the method of use.
0.001 to 10%.

As described above, the photosynthetic prokaryotic microorganism culture filtrate and / or the photosynthetic prokaryotic microorganism extract are added to the basic medium, and the plant tissue is cultured using the medium. The basic medium, the culture method, and the like are those used in ordinary plant tissue culture. Is the same as That is, as a basic medium, Murashig
e) & Skoog medium (1962) can be mentioned as a representative one, but various other media suitable for plant culture or their modified media can be appropriately selected and used. Further, plant hormones, coconut milk, casein hydrolyzate, yeast extract and the like used in ordinary culture may be added in accordance with the purpose.

In the present invention, the plant to be cultured is not particularly limited, and any plant can be applied. It is known that plants to be cultured have totipotency. Ie explants (whole plant or part thereof)
Can be cultured. Primary culture or subculture of the whole plant or a part thereof can also be cultured. In particular, a site containing a large amount of useful substances in a plant is preferable.

(Example) Hereinafter, an Example demonstrates in detail.

Example 1 The effects of adding a photosynthetic prokaryotic microorganism culture filtrate and / or a photosynthetic prokaryotic microorganism extract to a culture medium on carrot cultured carrot cultured cells were examined.

(1) Preparation of photosynthetic prokaryotic microorganism culture filtrate and photosynthetic prokaryotic microorganism extract Synechococcus (Synech
ococcus sp.) ATCC 27192 and Rhodopseudomonas blastica ATCC 33485. After the photosynthetic prokaryotic microorganism was cultured under culture conditions specified by the ATCC, the culture was centrifugally filtered to obtain a filtrate, which was concentrated 100-fold by an evaporator. This concentrated solution is desalted with a mosaic charged membrane desalter (Desalton DS-103: manufactured by Tosoh Corporation).
And filtered using a 0.45 μm membrane filter, and the resulting filtrate was used as the culture filtrate for photosynthetic prokaryotic microorganisms. The photosynthetic prokaryotic microbial extract is harvested, freeze-dried, and suspended in 3% of water.
Extraction with hot water at 60 ° C for 60 minutes, centrifugation and 0.45 μm of supernatant
It was obtained by filtration using a membrane filter.

(2) Culture of carotenoid-producing carrot cultured cells and measurement of carotenoid content Carotenoid-producing carrot cultured cells are cells having a carotenoid-producing ability derived from roots of ginseng.

The medium is Murashige & Skoog
A medium was used, and sucrose (3%) and 2,4-D (1 mg / l), which is a phytohormone of auxins, were added thereto.
An agar solid medium prepared at 5.5 to 5.7 and added with 1% agar was used as a basal medium. This basic medium was supplemented with 1% of a photosynthetic prokaryotic microorganism culture filtrate and a photosynthetic prokaryotic microorganism extract, and those without the addition were prepared, and carotenoid-producing carrot cultured cells were cultured at 25 ° C under dark conditions for 50 days. The amount of carotenoid accumulated in the cells was measured and compared. The amount of carotenoids was determined by measuring the fresh weight of the cultured cells, crushing the cells in a mortar, extracting with a small amount of acetone, adding 3 ml of petroleum ether, transferring the carotenoids into the petroleum ether layer, and using a spectrophotometer. Was used to measure the absorbance of the petroleum ether layer at 453 nm, and the amount of carotenoid accumulated in the cultured cells was calculated. Table 1 shows the results.

Example 2 The effect of adding a photosynthetic prokaryotic microorganism culture filtrate and / or a photosynthetic prokaryotic microorganism extract to a culture medium on the cultured cells of the pokeweed pokeweed producing betacyanin was examined. The cultured pokeweed cells are cells having a betacyanin-producing ability derived from stems of pokeweed.

The medium is Murashige & Skoog
Use medium, add sucrose (3%), 2,4-D 0.1mg
/ l was added thereto, and a medium adjusted to pH 5.5 to 5.7 was used as a basal medium. To the basic medium, 1% of the photosynthetic prokaryotic microorganism culture filtrate and / or photosynthetic prokaryotic microorganism extract prepared in Example 1 (1) was added, and one without the addition was prepared.
Under light irradiation (2000 lux) conditions, the cultured cells of betacyanine-producing pokeweed at 25 ° C. were subjected to liquid suspension culture for 14 days, and the amounts of betacyanin accumulated in the cells were measured and compared.

After measuring the fresh weight of the cultured cells, the cells were crushed in a mortar, extracted by adding a small amount of water, and after centrifugation, the absorbance of the supernatant at 535 nm was measured using a spectrophotometer. The amount of betacyanine accumulated in the cells was calculated. Table 2 shows the results.

(Effects of the Invention) According to the present invention, useful substances produced by plants are efficiently produced by culturing plants using a medium containing a photosynthetic prokaryotic microorganism extract and / or a photosynthetic prokaryotic microorganism culture filtrate. And can be a treatment method for minimizing the adverse effects of plant hormones.
Therefore, it is possible to improve the ability to produce useful substance-producing cells by tissue-culturing a plant. In addition, since it also has the effect of promoting the growth (proliferation) of useful substance-producing cells, a large amount of useful substance-producing cells can be rapidly obtained. Therefore, useful substances can be easily produced using the plant tissue culture technique.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hiroki Umezu 4-1-8 Yoshimachi, Soka City, Saitama Prefecture Pentel Inside the Grass Processing Plant (72) Inventor Isao Matsunaga 2-41-13 Fuchu, Fuchu-shi, Tokyo Fuchu Third House 2-304 Examiner Yoshiki Tanemura

Claims (2)

(57) [Claims] 1. A method for producing a useful substance produced by a plant by using a plant tissue culture technique, wherein a culture medium for plant tissue culture containing a filtrate for photosynthetic prokaryotic microorganism culture and / or an extract of photosynthetic prokaryotic microorganisms is used. Useful substance production method. 2. The method according to claim 1, wherein the photosynthetic prokaryotic microorganism is a cyanobacterium and / or a photosynthetic bacterium.